Dr. Katherine Jones (Salk Institute) has submitted a grant application that is a renewal of "Molecular Mechanisms of Gene Expression in Animal Cells" which is currently in the ninth year of funding. The proposed project aims to continue investigations on the molecular mechanism of transcription directed by the promoter in the HIV-1 long terminal repeat (LTR). Previous studies have largely utilized naked viral DNA templates, containing promoter elements, in reconstituted in vitro transcription systems to examine the roles of cellular and viral factors in initiation of RNA synthesis. To more accurately reflect the state of viral DNA in the nucleus of infected cells, this project will examine in vitro transcription from chromatin templates. Four specific aims are proposed to provide an understanding of the influence of chromatin on the enhancer, promoter (initiator), and Tat-responsive domain (TAR) in the HIV LTR. Specific Aim 1: To investigate the function of the viral enhancer, in vitro assays will be performed with chromatin templates and NF-kB (p65), and cellular co-activators interacting with p65 will be identified; additionally, the role of the cellular factor NF-AT in HIV transcription will also be examined. Specific Aim 2: To study the repressive effects of a nucleosome positioned on the viral promoter, the roles of LBP-1 and HIP-1 as well as other cellular factors will be analyzed. Specific Aim 3: To examine attenuation of HIV transcripts in the absence of Tat, formation of short viral transcripts will be monitored in vitro with naked DNA templates and chromatin. Specific Aim 4: To analyze Tat-mediated transcriptional transactivation, chromatin templates will be used in in vitro transcription systems with Tat; additionally, the requirement for exon-2 of Tat will be determined.